United States       Prevention, Pesticides     EPA712-C-96-343
          Environmental Protection    and Toxic Substances     February 1996
          Agency        (7101)
&EPA    Microbial Pesticide
          Test Guidelines
          OPPTS 885.4650
          Aquatic Invertebrate
          Range Testing, Tier

     This guideline is one  of a  series  of test  guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental  Protection Agency for use  in the testing of
pesticides and toxic substances, and the  development of test data that must
be submitted to the Agency  for review under Federal regulations.

     The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has  developed this guideline through  a process of harmonization that
blended the testing  guidance  and requirements that  existed in the Office
of Pollution Prevention and  Toxics  (OPPT) and appeared in Title  40,
Chapter I,  Subchapter R of the Code of Federal Regulations  (CFR),  the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical  Information Service (NTIS) and the guidelines pub-
lished by the Organization  for Economic Cooperation and Development

     The purpose of harmonizing these  guidelines  into a single set of
OPPTS guidelines is to minimize  variations among the testing procedures
that must be performed to meet the data  requirements of the U. S. Environ-
mental Protection Agency  under  the Toxic  Substances  Control Act  (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).

     Final  Guideline Release: This guideline  is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin   Board.   By  modem  dial   202-512-1387,  telnet   and   ftp:
fedbbs.access.gpo.gov    (IP,    internet:     http://
fedbbs.access.gpo.gov, or call 202-512-0132 for disks  or paper copies.
This guideline is also available electronically in ASCII and PDF (portable
document format) from the EPA Public Access Gopher  (gopher.epa.gov)
under the heading "Environmental Test  Methods and Guidelines."

OPPTS 885.4650  Aquatic invertebrate range testing, Tier III.
     (a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements  of the Federal Insecticide,  Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.}.

     (2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP guideline 153A-27.

     (b) Test standards. Data must be derived from tests that satisfy  the
general test standards in OPPTS 885.0001 and OPPTS 885.5000 through

     (1) Test organisms. Species most likely to be  affected by  the MPCA
should be used in this test. If a microorganism that is closely related (with-
in the same family) to the MPCA  causes disease in a certain invertebrate
species, that species should be included in this test.

     (2) Use pattern, (i) Microbial pest  control  agents that  are expected
to enter freshwater ecosystems should be tested on  freshwater  aquatic in-
vertebrates. Likewise, if an estuary is likely to be impacted, marine inver-
tebrates should be included.

     (ii) Testing should be performed on two members of the following

     Freshwater—Cladocera, Copepoda, Ephemeroptera, Trichoptera.

     Marine—Copepoda, Crustacea.

     (3) Method of pesticide administration. The test substance shall be
administered either  as a suspension in the test water  (aqueous exposure)
and/or in the diet as determined from results of Tier I tests.

     (4) Dose levels. The dose  level  shall be equal to that expected to
be found in the aquatic environment calculated from application rates with
appropriate  adjustments to take  into account the environmental survival
and multiplication characteristics of the MPCA being tested, as  determined
by Tier II testing.

     (5) Test duration. The test duration shall be the same as Tier I stud-

     (c) Reporting  and evaluation of data.  The requirements in Tier I,
OPPTS 885.4050  and 885.4100, apply.

     (d)  Tier  progression.  (1) Further testing   at Tier  IV  (OPPTS
885.4750) may be required if pathogenic effects are  observed,

     (2) Additional testing at higher tiers ordinarily is not required if path-
ogenic effects are not observed.

     (e) References. The following references should be consulted for ad-
ditional background material on this test guideline.

     (1) American Public Health Association. Standard Methods for Exam-
ination  of Water and Wastewater. 14th edition, Washington, DC  (1975).
pp. 1193

     (2) ASTM  Standard E 729-80, Practice for Conducting Acute Tox-
icity Tests with Fishes,  Macroinvertebrates, and Amphibians. American
Society for  Testing  and Materials, 1916  Race  Street,  Philadelphia, PA

     (3) Bioassay Procedures  for the  Ocean  Disposal  Permit Program.
USEPA, Office  of Research and Development.  EPA-600/9-78-010;pp.
121 (1978).

     (4) Banner,  L.H. et al. A salt-water  flow-through bioassay method
with controlled temperature and salinity, Progress in Fish-Culture 37:126-
129 (1975).

     (5) Clark, J.R. and R.L. Clark, eds. Seawater systems for experimental
aquariums. US Interior Department, Fish and Wildlife Service, Bureau of
Sport. Fish. Wild. Research Report No. 63,  192  pp. (1964).

     (6) Committee  on  Methods  for Toxicity  Tests  with Aquatic Orga-
nisms.  Methods  for acute toxicity tests with fish,  macroinvertebrates, and
amphibians.  USEPA Ecological Research  Series,  EPA  660/3-75-009.  61
pp. (Marine  and  estuarine species  listed in this publication are acceptable.)

     (7) Couch, J.A. et al. Environmental significance  of baculovirus infec-
tions in estuarine and  marine shrimp. Annals N.Y. Academy  of Science
219:528-536 (1975).

     (8) Couch, J.A.  Design and Test of a  Simple System for the Prelimi-
nary Evaluation  of Infectivity and Pathogenesis of Insect Virus in a Non-
target Estuarine  Shrimp. Journal of Invertebrate Pathology 43:351-357

     (9) DeBen, E.A. Design and construction of saltwater environment
simulator. Federal Water Quality Administration, Pacific N.W. Water Lab-
oratory, Working Paper 71:1-30 (1970).

     (10) Hetrick, P.M.  et al. Increased susceptibility of rainbow trout to
infectious hematopoetic necrosis virus after exposure to copper. Applied
Environmental Microbiology 37:198-201 (1979).

     (11)  Huang, E. and J.S. Pagano. Nucleic  acid hybridization  tech-
nology and detection of proviral genomes. Chapter 13 in The Atlas of In-
sect and Plant Viruses, K. Maramorosch, ed. Academic  Press, NY(1977).

     (12) Ignoffo,  C.  M. et al. Susceptibility of aquatic vertebrates  and
invertebrates to the infective stage of the mosquito nematode, Reesimermis
nielseni. Mosquito News 33:599-602 (1973).

     (13) Lightner, D.V. et  al.  Testing Penaeid shrimp  for susceptibility
to an insect Nuclear Polyhedrosis virus. Environmental Entomology 2:611-
613 (1973).

     (14) Pagano,  J.S.  and E. Huang. The application of  RNA-DNA
cytohybridization to   viral  diagnostics.  In:  Viral  Immunodiagnosis.  E.
Kurstak and R. Morisset, eds. Academic Press, NY (1974).

     (15) Reynolds,  G.J.  Enzyme  labelled  antibody in histopathology.
Qualityline Winter 1978/1979:2-10 (1978).

     (16)  Shelbourne, HE.  1962.  Experimental  seawater  systems  for
rearing  fish  larvae.  Pp.81-93  in  Seawater Systems  for  Experimental
Aquariums. J.R. Clark and R.L. Clark,  eds. U.S. Dept. Int.,  Fish. Wild.
Serv., Bur. Sport Fish. Wild. Res. Rep. No.63. 192 pp.

     (17) Strickland, J.D.H., and T.R. Parsons. 1968. A practical handbook
of seawater analysis. Fish Res. Board Can. Bull. No. 167., 311 pp.

     (18)  Summers,   M.,  R. Engler, L.A.  Falcon,  and P. Vail, eds.
Baculoviruses for Insect Pest Control: Safety Considerations. Selected pa-
pers from EPA-USDA Working Symposium, American Society for Micro-
biology, Washington, DC (1975).

     (19)  Tamer,  M.F.  et  al.  The  tissue localization of  Aeromonas
salmonicida in rainbow trout, Salmo gairdneri Richardson, following three
methods of administration. Journal of Fish Biology 25:95-108 (1984).

     (20) Undeen A.H.  and J.V. Maddox. The infection of nonmosquito
hosts by injection with spores of the microsporidan Nosema algerae. Jour-
nal of Invertebrate Pathology 22:258-265 (1973).

     (21) Van Essen, F.W. and D.W. Anthony. Susceptibility of nontarget
organisms to  Nosema Algerae (Microsporida: Nosematidae), a parasite of
mosquitoes. Journal Invertebrate Pathology 28:77-85 (1976).

     (22) Weber, C.E. (ed.) Biological field laboratory methods for meas-
uring the quality of surface waters  and  effluents. USEPA Environmental
Monitoring Series, EPA-670/473-001 (1973).